TOPICAL EXOSOME COMPOSITIONS AND ASSOCIATED METHODS

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of, and priority to, U.S. Provisional Patent Application Serial No. 62/877, 155, filed July 22, 2019, the entirety of which is incorporated by reference and commonly owned.

FIELD OF THE INVENTION

[0002] The present disclosure relates generally to topical exosome compositions for use in treating a variety of conditions in a subject, as well as methods of making and using the same.

BACKGROUND

[0003] Extracellular vesicles (EVs) including exosomes, microvesicles, and apoptotic bodies are nanoscale spherical vesicles that are released when the multivesicular bodies (MVBs) fuse with the cell plasma membrane. Consequently, they bud and are shed into the extracellular space and bodily fluids. Exosomes are increasingly important in cell biology because of their role in cell-to- cell communication and transfer of cellular materials, such as miRNA, IncRNA, mRNA, proteins, and DNA. This mechanism implies that exosomes underlie some illnesses such as cancer, neurological, cardiovascular, and rheumatologic diseases. Different cell types grown in vitro constantly and constitutively secrete exosomes into the culture media. Therefore, the conditioned culture medium of cells serves as the very food for exosome manufacturing. Notably, many biopharmaceutical companies are already producing the exosomes during the cell culture processes, but these exosomes are discarded as a waste by-product. Exosomes, however, can be applied in different translational paradigms comprising diagnostics, therapeutics, and research. The embodiments disclosed herein are aimed at fulfilling these and other needs in the art.

SUMMARY

[0004] The present disclosure relates to novel topical exosome compositions and methods of making and using the same. Exemplary compositions disclosed herein include (1) a hair loss solution comprising minoxidil, hydroxypropyl-b-cyclodextrin (HPbCD), milk exosomes including peptide/protein fractions, PEG 6000 solution, and any combination thereof; (2) a skin lightening moisturizing cream for normal to dry skin comprising hydroquinone, Vitamins A (retinol) and B3, arbutin, kojic acid, sun protection factor (SPF) and any combination thereof; (3) an anti-wrinkle eye cream comprising milk exosomes, vanishing cream, ferulic acid, hyaluronic acid, retinol, and any combination thereof; (4) a rejuvenating eye cream comprising mesenchymal stem cells (MSC) exosome, secretome, and any combination thereof; (5) a facial skin rejuvenation cream comprising MSC and Milk exosomes, and any combination thereof; (6) an anti-aging composition (antioxidants, aka Serum) comprising retinols, milk exosomes and peptides (collagen stimulants), A-MSC exosomes under hyperbaric O ₂ , and any combination thereof; (7) a wound healing cream comprising platelet-rich plasma (PRP), induced pluripotent stem cells (iPCs), MSC exosomes, oil in water (O/W) cream, and any combination thereof; (8) a diabetic wound treatment comprising Hyperbaric CO ₂ induced MSC exosomes (angiogenic factors; overexpressing Nrf2), and any combination thereof; (9) an atopic dermatitis treatment comprising A-MSC exosomes and any combination thereof; (10) a moisturizing day/night lip cream or balm comprising rejuvenating and antioxidant factors such as exosomes and any combination thereof; (11) a skin tanning moisturizing cream for normal to dry skin comprising dihydroxyacetone (DHA), Vitamins A (retinol) and/or B3 and any combination thereof; (12) a facial/eye mask comprising rejuvenating and antioxidant factors such as exosomes and any combination thereof; and (13) an Acne Rosacea treatment comprising exosomes loaded with Azelaic acid, Isotretinoin and/or Retinoic acid and any combination thereof. The exemplary embodiments disclosed herein in connection with particular uses are not necessarily limited to said use(s), and multiple uses for the disclosed compositions are envisioned and within the scope of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0005] For a fuller understanding of the embodiments disclosed herein, reference is made to the following detailed description, taken in connection with the accompanying drawings illustrating various embodiments of the present disclosure, in which:

[0006] FIG. 1A depicts western blot chemiluminescence curves for exosomes prepared in accordance with the teachings of the present disclosure; [0007] FIG. 1B depicts a quantitative analysis of the chemiluminescence curves of FIG. 1 A;

[0008] FIG. 1C depicts the western blots used to generate the data of FIGS. 1 A and 1B;

[0009] FIG. 2A depicts western blot chemiluminescence curves for exosomes prepared in accordance with the teachings of the present disclosure;

[0010] FIG. 2B depicts a quantitative analysis of the chemiluminescence curves of FIG. 2 A;

[0011] FIG. 2C depicts the western blots used to generate the data of FIGS. 2A and 2B;

[0012] FIG. 3A depicts western blot chemiluminescence curves for exosomes prepared in accordance with the teachings of the present disclosure;

[0013] FIG. 3B depicts a quantitative analysis of the chemiluminescence curves of FIG. 3A;

[0014] FIG. 3C depicts the western blots used to generate the data of FIGS. 3A and 3B;

[0015] FIG. 4A depicts western blot chemiluminescence curves for exosomes prepared in accordance with the teachings of the present disclosure;

[0016] FIG. 4B depicts a quantitative analysis of the chemiluminescence curves of FIG. 4 A;

[0017] FIG. 4C depicts the western blots used to generate the data of FIGS. 4A and 4B;

[0018] FIG. 5 A depicts the results of an MTT Assay after treatment of BJ fibroblasts with A-

MSC exosomes and secretome prepared in accordance with the teachings of the present disclosure;

[0019] FIG. 5B depicts the results of an MTT Assay after treatment of HEK293 cells with A- MSC exosomes and secretome prepared in accordance with the teachings of the present disclosure;

[0020] FIG. 6A depicts the results of a WST Assay 24 hours after treatment of BJ fibroblasts with A-MSC exosomes and secretome prepared in accordance with the teachings of the present disclosure;

[0021] FIG. 6B depicts the results of a WST Assay 24 hours after treatment of HEK293 cells with A-MSC exosomes and secretome prepared in accordance with the teachings of the present disclosure; [0022] FIG. 6C depicts the results of a WST Assay 48 hours after treatment of BJ fibroblasts with A-MSC exosomes and secretome prepared in accordance with the teachings of the present disclosure;

[0023] FIG. 6D depicts the results of a WST Assay 48 hours after treatment of HEK293 cells with A-MSC exosomes and secretome prepared in accordance with the teachings of the present disclosure; and

[0024] FIG. 7 depicts the results of a scratch assay on BJ fibroblasts treated with A-MSC exosomes and secretome.

DETAILED DESCRIPTION OF EMBODIMENTS

[0025] The present disclosure relates to exosome-based topical compositions and methods for making and using the same, including using the compositions for the treatment of a variety of conditions. It is to be understood that the descriptions of the present embodiments have been simplified to illustrate elements that are relevant for a clear understanding, while eliminating, for the purposes of clarity, many other elements that may be found in the present embodiments. Those of ordinary skill in the pertinent art will recognize that other elements are desirable and/or required in order to implement the present embodiments. However, because such elements are well known in the art, and because such elements do not facilitate a better understanding of the present embodiments, a discussion of such elements is not provided herein. Exosomes employed in connection with embodiments of the present disclosure may be bovine milk derived, MSC exosomes, A-MSC exosomes, HEK293 exosomes, NSC exosomes or any other exosome type suitable for the delivery purposes described herein that may be loaded by different methods or used as their natural form. The exosomes may be prepared in accordance with existing techniques known in the art. Alternatively, the exosomes may be prepared in accordance with one or more of the methods disclosed herein. In one or more embodiments, exosomes may be loaded with drugs such as, for example, minoxidil, hydroquinone, corticosteroids, or biologicals or natural compounds drug-excipient complexes. Drug may mean any biological or chemical active pharmaceutical ingredient or natural product or new candidate. Drug delivery may target skin layers such as epidermis, dermis or hypodermis or intended and employed for systemic drug delivery. Dosage forms for any one of the embodiments disclosed herein may include, but are not limited to, ointment, cream, gel, topical spray, patch, emulsion, suspension, solution and/or powder. Other pharmaceutical dosage forms may also be used to deliver drugs including but not limited to injectables for dermal drug delivery.

[0026] Reference throughout this specification to“one embodiment”;“an embodiment”;“one or more embodiments”; or the like means that a particular feature, structure, step, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases “in one embodiment”; “in an embodiment”; “one or more embodiments”; or the like in various places throughout this specification are not necessarily all referring to the same embodiment. Moreover, the individual elements of a composition or steps in a method may be interchanged between the various exemplary embodiments without departing from the scope of the present disclosure.

[0027] In addition, for the purpose of promoting an understanding of the principles of the present disclosure, reference will now be made to the embodiments described herein, and specific language will be used to describe the same. It will, nevertheless, be understood that no limitation of the scope of the disclosure is thereby intended, and any alterations and further modifications of the described or illustrated embodiments and any further applications of the principles of the disclosure as illustrated herein are contemplated as would normally occur to one skilled in the art to which the disclosure relates. All limitations of scope should be determined in accordance with and as expressed in the eventual claims of one or more issued patents.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present embodiments, exemplary preferred methods and materials are described.

[0029] The articles“a” and“an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element. [0030] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, encompasses variations of ±20%, or ±10%, or ±5%, or ±1%, or ±0.1% from the specified value, as such variations are appropriate to prepare the disclosed compositions and perform the disclosed methods.

[0031] Throughout this disclosure, various aspects of the embodiments are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the embodiments. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 2, 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2-5, from 2 to 6, from 3 to 6 and so on, as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0032] The following techniques may be employed in connection with one or more embodiments of the present disclosure. One of ordinary skill in the art, having the benefit of the present disclosure, will appreciate that other known techniques may be employed without departing from the spirit and scope of the present disclosure.

[0033] Exemplary Method for Preparing Exosomes

[0034] While at least one exemplary protocol for the manufacture of exosomes is provided herein, one of ordinary skill in the art, having the benefit of the present disclosure, will appreciate that alternative methods for purifying and/or preparing exosomes may be employed without departing from the scope of the present disclosure. Accordingly, the compositions and methods disclosed herein are not necessarily limited to use of the disclosed methods for preparing exosomes.

[0035] By way of non-limiting example, A-MSC cultures may be scaled from 1 liter to 5 liters and then 30 liters. We evaluate both fed batch and multiple conditioning run methods of culture at 30 liters. The yield is optimized using the final exosomal yield (EVs/ml of conditioned medium) conforming to reference standard criteria. The conformance is checked against release criteria (Table 1). The criteria for success include the quantitative milestones listed below. EVs are purified from the 30 liters of conditioned media using filtration followed by multimodal chromatography. We typically produce at least 5E13 EVs per liter of conditioned media following nutrient supplement deprivation. However, other stressing regimens such as oxidative stress or chemicals are also employed.

[0036] The following quantitative milestones are assessed. (1) Culture 30 liters of A-MSC cells on microcarriers in bioreactor. The bioreactors may include stirred tank, rocking bed bioreactor, wave bioreactor, packed bed bioreactor, circulation bioreactor or vertical wheel bioreactor. EVs are purified using filtration with the following criteria: (2) Produce EVs with 90% having a size of 100 nm (± 80-120 nm). (3) Generate yields of at least 5E13 EVs per L of conditioned media, (4) Contain protein not greater than 12.5 ug protein per ug of EVs, (5) Identify the presence of a lipid bilayer in the EVs using electron microscopy, (6) Detectable presence of a minimum of three of the following EV-specific proteins (CD9, CD63, CD81, TSG101 and Annexin Al) by Western blot (lacking Calreticulin and GAPDH) and (7) Batch to batch variation of not more that 10% based upon the technical feedback from the center for biologies research and evaluation at the FDA.

[0037] To purify the exosomes in the downstream processing, methods employed include ways to isolate and purify EVs using spiral column filtration, multimodal column chromatography, viral deactivation and polishing rather than the traditional ultracentrifugation process. Spiral column filtration using 500-750 KDa cutoffs is used to concentrate and diafiltrate the conditioned medium. The retentate is chromatographed against anion exchange and size exclusion columns or a bimodal column. Column packing and eluent properties are optimized to improve the yield. The filtrate is filtered through 3-30 KDa dead-end or hollow fiber filter to concentrate the secretome to process further to isolate the growth factors. Acidic pH adjustment (~ 2-3.5) is used to deactivate the viruses. Further diafiltration against PBS is included to gain the final product concentration.

Table 1. Exemplary Quality Control and Release Criteria.

[0038] 30 liters of culture media of A-MSCs containing at least 1E13 of EV/ L is collected and subjected to in-depth filtration series 25 mm, 10 mm, 4 mm, 1 mm, 0.22 mm. The filtrate is

5U B5TITUTE SHEET (RULE 26) concentrated to 600 ml using hollow fiber cartridge filtration (GE) with 500-750 KDa cut-off filters. In order to remove DNA, the concentrated sample (50X) is treated with Benzonase endonuclease. Benzonase endonuclease degrades all nucleic acids down to oligonucleotides of approximately 3 to 5 base pairs in length. These small fragments are removed by next step diafiltration and also can enter into the core of chromatography system where they are bound by the octylamine ligands. The Benzonase endonuclease itself can also be removed by chromatography system. Then, the sample is subjected to 10-times diafiltration using PBS. The diafiltrated sample is chromatographed against a multimodal chromatography resin. 300 ml of resin is packed in a column and equilibrated with PBS. The EV-rich sample (600 ml) is loaded onto the column at a flow rate of 20 ml/min. After running the sample, the column is washed with 300 ml of PBS buffer and then with 0.1M NaOH in 27% 1-propanol as cleaning in place (CIP) solution. The pH of CIP fractions are immediately adjusted to 7.5 with 0.1M HC1 in order to analyze the bound proteins. Chromatography column is regenerated with 1M NaOH in 27% 1- propanol solution. 10 ml fractions are collected at each sample loading step to evaluate purified EV and also at elution step to analysis of bounded proteins. Particle size and concentration of EVs are determined via nanoparticle tracking analysis (NT A). Protein quantities in samples are measured using bicinchoninic acid assay kit and RNA concentration are quantified using the RNA assay kit. Western blots are performed in order to test for the presence of surface markers CD9, CD63, CD81, TSG101 and Annexin Al . Image analysis is performed using Image J software. Acidic pH adjustment (3.5) for 1 hour is used to deactivate the viruses. Further diafiltration against PBS is included to gain the final product concentration on a hollow fiber or spiral column filter system.

[0039] Exosomes may be lyophilized using cryoprotectants and stabilizers such as proteins (peptides), cyclodextrin, or sugars. Process parameters are temperature (deep freeze, -50 to -85°C), vacuum (50-200 mTorr), and drying time (36-72 h) depending on the solution buffer and exosome concentration.

[0040] Second Exemplary Method for Preparing Exosomes

[0041] A-MSC cells are cultured in T-25, T-75 flasks, then transferred to 100 ml bioreactor on Cytodex 1 or 3 microcarriers. The cells are cultured for 7 days in 250 ml and 500 ml bioreactors and conditioned for 2 days. The conditioning regimen included replacing the media with the basal media and removing any supplements and growth factors. After media harvest, the conditioned medium was filtered through 20 um, 10 um, 4 um and 0.22 um filters. The cleared conditioned media was (ultra)-centrifuged at the following: 300g for 10 min; 2000g for 20 min; 10,000g for 30 min; and 100,000g for 4 h or 120,000g for 2 h or 150,000g for lh. Then the pellet is resuspended in PBS for storage at -80oC. Alternatively, 750 KDa filtration could be used to prepare the exosomes crude samples and then run the ultracentrifugation step or run Capto Core multimodal chromatography.

[0042] Exemplary Method of Secretome Preparation.

[0043] The method for exosome preparation was used with added steps for secretome preparation. The supernatant after ultracentrifugation was passed through 3 KDa filter and the retentate was collected. The concentration ratio ranged from 35 to 50 times the initial volume.

[0044] Exemplary Methods for Loading of Exosomes with Drugs or Other Components

[0045] The loading of exosomes with drugs can be achieved with various known methods. The choice of method is often dependent on the physical properties of the drug of interest. For instance, a drug with an overall negative charge may be very difficult to load into exosomes using a passive method because exosomes carry an overall negative charge.

[0046] Therefore, one or more embodiments of the present disclosure may utilize electroporation to load exosomes with various chemical components. By way of non-limiting example, an electroporation method may be used to load drug(s) or other components into exosomes, but in order to do so, exosomes need to be previously reconstituted and frozen away in Optiprep-free (see recipe below) electroporation buffer. Large numbers (e.g., 5 x 10 ⁹ to 5 x 10 ¹⁰ ) of exosomes are brought to 1X Optiprep using the 2X Optiprep buffer, and the total reaction mix is brought up to 400 ul with drug and 1X Optiprep Electroporation buffer. Note that, in one or mpore embodiments, the required drug volume should be as low as possible such that the buffer is not compromised; if drug volume is excessive, consider using 2X buffer to compensate. [0047] Exemplary Electroporation Buffer Recipe:

Components

3M KC1 (Mettler Toledo # 63056166), 0.22 urn filtered

0.5 M Potassium Phosphate, pH 7.2 (Alfa Aesar # J60326), 0.22 um filtered

OptiPrep (Stemcell Technologies # 07820), sterile

Water (Hyclone Molecular Biology Grade), sterile

Electroporation Buffer

57.5 ul 0.5M K-Phosphate

208.3 ul 3M KC1

5.25 ml OptiPrep

19.45 ml Water

[0048] Note that this is 1X OptiPrep. When used to reconstitute EVs, one would have to do so with buffer without OptiPrep (OptiPrep should not be frozen); 2X OptiPrep would be required when using your frozen EVs such that the final OptiPrep concentration is 1X in your electroporation buffer. Exemplary electroporation reaction conditions (400 ul total volume) comprise 0.4 cm electroporation cuvettes, 400V, 125 uF.

[0049] An alternative exemplary method for loading exosomes comprises an incubation protocol. Incubation is a passive method of loading drug into exosomes. Simply, drug and exosomes are suspended together and incubated until loading is maximized. This works well for neutral or positively charged drugs. We have successfully loaded exosomes suspended in DPBS with drug at various temperatures (37 °C and 4 °C, for example). Also, loading time may vary, and we have used loading times as low as 5 minutes, and loading times of 18 hours.

[0050] Yet another exemplary exosome loading protocol may utilize commercially available reagents such as Lipofectamine (Therm oFisher) and Exo-Fect (SBI), which are practical options to use in cases where exosome loading is challenging. Size and/or charge on drugs often makes exosome loading difficult, so using commercial reagents should be kept in mind in such cases.

[0051] Other exemplary loading methods are available and can be viable options. Methods such as sonication and extrusion are methods used with success. Also, combinations of methods may be utilized, such as, for example, using commercial reagents combined with electroporation. [0052] Exemplary Embodiment 1

[0053] One or more embodiments of the present disclosure provide a composition for treating hair loss comprising minoxidil; hydroxypropyl-b-cyclodextrin (HPbCD); milk exosomes including peptide/protein fractions; and MC, PG, PEG 6000 and/or any combination thereof. In one or more exemplary embodiments, the composition comprises about 5% minoxidil; about 2% hydroxypropyl-b-cyclodextrin (HPbCD; milk exosomes including peptide/protein fractions); (MC, PG, PEG 6000); and any combination thereof. Alternatively, one or more exemplary embodiments may comprise minoxidil powder USP; ethanol absolute; hydroxypropyl-b- cyclodextrin (HPbCD) pharmaceutical grade; propylene glycol (preservative; stabilizing agent; cosolvent); glycerin; DL-Lactic acid; bovine raw milk exosomes; bovine raw milk ultracentrifugation peptide/protein fraction and/or 750 KDa TFF filtrate (100 KDa retentate); MSC exosomes; MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate); methyl cellulose (MC); and/or Polyethylene Glycol 6000 and any combination thereof.

[0054] By way of non-limiting example, one or more exemplary embodiments of the present disclosure comprise 5g of minoxidil powder USP; 1-5ml of Ethanol absolute; 20-35g of hydroxypropyl-b-cyclodextrin (HPbCD) pharmaceutical grade; 5-10ml of propylene glycol (Preservative; stabilizing agent; cosolvent); 5-20ml of glycerin; 2.5-5ml of DL-Lactic acid; 1ml of bovine raw milk exosomes (1E12 EVs/ml); 100ml of bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate); MSC exosomes (1E12 EVs/ml); MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate); methyl cellulose (MC) (1-2%); polyethylene glycol 6000 (up to 10% W/V) to achieve viscosity of 15-25 cP; and any combination thereof.

[0055] Exemplary methods for preparing the disclosed hair loss treatment compositions are herein provided. One of ordinary skill in the art, having the benefit of the present disclosure, will appreciate that alternative methods may be available without departing from the spirit and scope of the present disclosure. [0056] Exemplary Method 1.1

[0057] One or more embodiments of the present disclosure comprise at least one of (i) dissolving 20-35g of hydroxypropyl-b-cyclodextrin (HPbCD) in 75ml of WFI water and mixing at 70 rpm for 5 min at room temperature; (ii) dispersing 5g of minoxidil powder in 5ml ethanol (70%) on stirring at 100 rpm, wherein when the minoxidil is dissolved; (iii) adding the solution to 10ml of bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) on stirring at 100 rpm at room temperature; (iv) mixing the solution for 15 min at 100 rpm; (v) cooling the sample to about 8°C; (vi) adding 1ml of bovine raw milk exosomes (1E12 EVs/ml) on stirring at 45 rpm at about 4-8°C; (vii) adding bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) up to a total volume of 100ml; and (viii) adding methyl cellulose (MC) (1-2%) or polyethylene glycol 6000 (up to 10% W/V) to achieve a viscosity of aboutl5-25 cP.

[0058] Exemplary Method 1.2

[0059] One or more embodiments of the present disclosure comprise at least one of (i) dissolving 20-35g of hydroxypropyl-b-cyclodextrin (HRbOϋ) in 75ml of bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) and mixing at 70 rpm for 5 min at room temperature (mixture I); (ii) dissolving 5g of minoxidil powder in 5ml ethanol (70%) on stirring at 100 rpm at room temperature; (iii) adding 5ml of propylene glycol and continuing mixing, wherein when minoxidil is dissolved; (iv) adding the solution to mixture I on stirring at 100 rpm at room temperature; (v) mixing the solution for 15 min at 100 rpm; (vi) adding 1ml of bovine raw milk exosomes (1E12 EVs/ml) on stirring at 45 rpm at 16°C; (vii) adding bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) up to a total volume of 100ml; and (viii) adding methyl cellulose (MC) (1- 2%) or polyethylene glycol 6000 (up to 10% W/V) to achieve viscosity of 15-25 cP.

[0060] Exemplary Method 1.3

[0061] One or more embodiments of the present disclosure comprise at least one of (i) dissolving 20-35g of hydroxypropyl-b-cyclodextrin (HPbCD) in 75ml of bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) and mixing at 70rpm for 5 min at room temperature (mixture I); (ii) dissolving 5g of Minoxidil powder in 5ml ethanol (70%) on stirring at 100 rpm at room temperature; (iii) adding 5ml of glycerin and continuing mixing (mixture II) (iv) mixing mixtures I and II for 15 min at 50 rpm; (v) adding 1ml of bovine raw milk exosomes (1E12 EVs/ml) on stirring at 45 rpm in 16°C; (vi) adding bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) up to a total volume of 100ml; and (vii) adding methyl cellulose (MC) (1-2%) or polyethylene glycol 6000 (up to 10% W/V) to achieve viscosity of 15-25 cP.

[0062] Exemplary Method 1.4

[0063] One or more embodiments of the present disclosure comprise at least one of (i) dissolving 20-35g of hydroxypropyl-b-cyclodextrin (HPbCD) in 75ml of bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) and mixing at 70 rpm for 5 min at room temperature (mixture I); (ii) dissolving 5g of minoxidil powder in 5ml ethanol (100%) on stirring at 100 rpm at room temperature; (iii) adding 5ml of glycerin and continuing mixing; (iv) adding 2.5ml of Lactic acid and continuing mixing (mixture II); (v) mixing solutions I and II for 15 min at 50 rpm; (vi) adding 1ml of bovine raw milk exosomes (1E12 EVs/ml) on stirring at 45 rpm in 16°C; adding 1ml of MSC exosomes (1E12 EVs/ml) on stirring at 45 rpm in 16°C; (vii) adding bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) up to a total volume of 100ml; and (viii) adding methyl cellulose (MC) (1-2%) or polyethylene glycol 6000 (up to 10% W/V) to achieve a viscosity of 15- 25 cP.

[0064] Exemplary Method 1.5 (alcohol and propylene glycol free formulation)

[0065] One or more embodiments of the present disclosure comprise at least one of (i) dissolving 20-35g of hydroxypropyl-b-cyclodextrin (HPbCD) in 75ml of bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) and mixing at 70 rpm for 5 min at room temperature (mixture I); (ii) dissolving 5g of minoxidil powder in mixture I on stirring at 100 rpm at room temperature for 6h; (iii) adding 5ml of glycerin and continuing mixing; (iv) adding 1ml of bovine raw milk exosomes (1E12 EVs/ml) on stirring at 45 rpm in 16°C; (v) adding 1ml of MSC exosomes (1E12 EVs/ml) on stirring at 45 rpm in 16°C; (vi) adding 15ml of MSC secretome (750 KDa filtrate - 3 KDa retentate) on stirring at 45 rpm at 16°C; (vii) adding bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) up to a total volume of 100ml; and (viii) adding methyl cellulose (MC) (1- 2%) or polyethylene glycol 6000 (up to 10% W/V) to achieve viscosity of 15-25 cP.

[0066] Exemplary Embodiment 2

[0067] One or more embodiments of the present disclosure provide a composition and/or cream for skin lightening (brightening or whitening) comprising hydroquinone; vitamin A (retinol); vitamin B3; arbutin; kojic acid; and/or viola tricolor ethanolic extract and any combination thereof. In one or more exemplary embodiments, the composition comprises about 4% hydroquinone; vitamin A (retinol); vitamin B3; arbutin; kojic acid; and/or sun protection factor (SPF) and any combination thereof. Alternatively, one or more embodiments of the present disclosure may comprise O/W base cream (such as, for example, Humco PENcream™, Humco MultiBase™, Humco SaltStable LO™); Hydroquinone; Vitamin A (as retinol); Vitamin B3; Arbutin; Kojic acid as Kojic dipalmitate; bovine raw milk exosomes (1E12 EVs/ml); bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate); MSC exosomes; MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate); and any combination thereof.

[0068] By way of non-limiting example, one or more exemplary embodiments of the present disclosure comprise O/W base cream (such as, for example, Humco PENcream™, Humco MultiBase™, Humco SaltStable LO™); 2.4g of Hydroquinone; Vitamin A (as retinol) 2.5g/100g; Vitamin B3 5g/100g; Arbutin as 5g/100g; Kojic acid as Kojic dipalmitate 0.2g/100g; 0.6 ml of bovine raw milk exosomes (1E12 EVs/ml); 5ml of bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) 50X concentrate; 0.6ml of MSC exosomes (1E12 EVs/ml); and/or 1ml of MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate and any combination thereof.

[0069] Exemplary methods for preparing the disclosed skin-lightening compositions are herein provided. One of ordinary skill in the art, having the benefit of the present disclosure, will appreciate that alternative methods may be available without departing from the spirit and scope of the present disclosure. [0070] Exemplary Method 2.1

[0071] One or more embodiments of the present disclosure comprise at least one of (i) selecting an O/W base cream from a commercial source; (ii) providing 30g of the base cream at ambient temperature; (iii) dissolving 2.4g of hydroquinone in 3ml of alcohol; (iv) adding the solution to 30g of base cream and mixing at 50 rpm in 45°C for 15 min; (v) adding Vitamin A (as retinol) 2.5g/100g, Vitamin B3 5g/100g, Arbutin 5g/100g, and Kojic acid as Kojic dipalmitate 0.2g/100g and mixing at 50 rpm at 45°C for 10 min; (vi) adding 0.6 ml of bovine raw milk exosomes (1E12 EVs/ml) and 5ml of bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) 50X concentrate; (vii) mixing at 45 rpm at 20°C for 10 min; and (viii) adjusting to 60g using base cream.

[0072] Exemplary Method 2 2

[0073] One or more embodiments of the present disclosure comprise at least one of (i) selecting an O/W base cream from commercial source; (ii) providing 30g of the base cream at ambient temperature; (iii) dissolving 2.4g of hydroquinone in 3ml of alcohol; (iv) adding the solution to 30g of base cream and mixing at 50 rpm at 45°C for 15 min; (v) adding Vitamin A (as retinol) 2.5g/100g, Vitamin B3 5g/100g, Arbutin 5g/100g, and Kojic acid as Kojic dipalmitate 0.2g/100g and mixing at 50 rpm in 45°C for 10 min; (vi) adding 0.6ml of bovine raw milk exosomes (1E12 EVs/ml), 2.5ml of bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) 50X concentrate, 0.6ml of MSC exosomes (1E12 EVs/ml), and 1ml of MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate and mixing at 45 rpm at 20°C for 10 min; and (vii) adjusting to 60g using base cream.

[0074] Exemplary Method 2 3

[0075] One or more embodiments of the present disclosure comprise at least one of (i) selecting an O/W base cream from commercial source; (ii) providing 30g of the base cream at ambient temperature; (iii) dissolving Viola tricolor ethanolic extract in appropriate solvent and adding to the base; (iv) adding the solution to 30g of base cream and mixing at 50 rpm at 45°C for 15 min; (v) adding Vitamin A (as retinol) 2.5g/100g and Vitamin B3 5g/100g and mixing at 50 rpm at 45°C for 10 min; (vi) adding 0.6 ml of bovine raw milk exosomes (1E12 EVs/ml), 5ml of bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) 50X concentrate, 0.6ml of MSC exosomes (1E12 EVs/ml), and 1ml of MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate and mixing at 45 rpm at 20°C for 10 min; and (vii) adjusting to 60g using base cream.

[0076] Exemplary Embodiment 3

[0077] One or more embodiments of the present disclosure provide a composition and/or cream for eye rejuvenation comprising MSC exosomes and secretome. In one or more exemplary embodiments, a composition comprises a vanishing base cream (supplied from commercial source; such as Humco PENcream™ or Humco MultiBase™); Vitamin A (as retinol); Vitamin B3; Vitamin E; Hydrolyzed collagen + hyaluronic acid (Cromoist HTA); Jojoba oil; Allantoin; Fibronectin; bovine raw milk exosomes; bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate); MSC exosomes; and MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) and any combination thereof. In one or more embodiments, the MSC exosomes comprise A-MSC exosomes.

[0078] In one or more exemplary embodiments, a composition comprises a vanishing base cream (supplied from commercial source; such as Humco PENcream™ or Humco MultiBase™); Vitamin A (as retinol) 2.5g/100g; Vitamin B3 5 g/100g; Vitamin E 50,000IU/100g; Hydrolyzed collagen + hyaluronic acid (Cromoist HTA) 2g/100g; Jojoba oil 2g/100g; Allantoin 0.5g/100g; Fibronectin 0.5g/100g; 0.5 ml of bovine raw milk exosomes (1E12 EVs/ml); 0.5ml of bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) 50X concentrate; 1ml of MSC exosomes (1E12 EVs/ml); and 1ml of MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate and any combination thereof.

[0079] Exemplary methods for preparing the disclosed eye-rejuvenating compositions are herein provided. One of ordinary skill in the art, having the benefit of the present disclosure, will appreciate that alternative methods may be available without departing from the spirit and scope of the present disclosure. [0080] Exemplary Method 3.1

[0081] One or more embodiments of the present disclosure comprise at least one of (i) selecting a vanishing base cream from commercial source such as Humco PENcream™ or Humco MultiBase™; (ii) providing 5g of the base cream at ambient temperature; (iii) adding Vitamin A (as retinol) 0.25g/10g, Vitamin B3 0.5 g/10g, E 5,000IU/10g, Hydrolyzed collagen + hyaluronic acid (Cromoist HTA) 0.2g/10g and mixing at 50 rpm at 45°C for 10 min; (iv) adding 0.5 ml of bovine raw milk exosomes (1E12 EVs/ml), 0.5ml of bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) 50X concentrate, 1ml of MSC exosomes (1E12 EVs/ml), and 1ml of MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate and mixing at 45 rpm at 20°C for 10 min; and (v) adjusting to 10g using base cream.

[0082] Exemplary Method 3 2

[0083] One or more embodiments of the present disclosure comprise at least one of (i) selecting a vanishing base cream from commercial source such as Humco PENcream™ or Humco MultiBase™; (ii) providing 5g of the base cream at ambient temperature; (iii) adding Vitamin A (as retinol) 0.25g/10g, Vitamin B3 0.5 g/10g, E 5,000IU/10g, Hydrolyzed collagen + hyaluronic acid (Cromoist HTA) 0.2g/10g and mixing at 50 rpm at 45°C for 10 min; (iv) adding Jojoba oil 0.2g/10g, Allantoin 0.05g/10g, and Fibronectin 0.05g/10g; (v) adding (adjust concentrations and amounts as per need to make the formulation work) 0.5 ml of bovine raw milk exosomes (1E12 EVs/ml), 0.5ml of bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) 50X concentrate, 1ml of MSC exosomes (1E11 EVs/ml), 1ml of MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate and mixing at 45 rpm at 20°C for 10 min; and (vi) adjusting to 10g using base cream.

[0084] Exemplary Method 3 3

[0085] One or more embodiments of the present disclosure comprise at least one of (i) selecting a vanishing base cream from commercial source such as Humco PENcream™ or Humco MultiBase™; (ii) providing 5g of the base cream at ambient temperature; (iii) adding Vitamin A (as retinol) 0.25g/10g, Vitamin B3 0.5 g/10g, E 5,000IU/10g, Hydrolyzed collagen + hyaluronic acid (Cromoist HTA) 0.2g/10g and mixing at 50 rpm in 45°C for 10 min; (iv) adding Jojoba oil 0.2g/10g, Allantoin 0.05g/10g, and Fibronectin 0.05g/10g; (v) adding 1ml of MSC exosomes (1E12 EVs/ml) and 1ml of MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate and mixing at 45 rpm at 20°C for 10 min; and (vi) adjusting to 10g using base cream.

[0086] Exemplary Embodiment 4

[0087] One or more embodiments of the present disclosure provide a composition for at least one of an anti-wrinkle and brightening eye serum comprising milk exosomes; vitamin C; ferulic acid; hyaluronic acid; Retinol; and any combination thereof. One or more embodiments of a composition may also comprise a vanishing base cream (supplied from commercial source; such as Humco PENcream™ or Humco MultiBase™); Vitamin A (as retinol); Vitamin B3; Vitamin E; Vitamin C (as Tetrahexyldecyl Ascorbate (THD Ascorbate); Magnesium ascorbyl phosphate (MAP); Ascorbyl 6 palmitate; Disodium isostearyl 2-0 L-ascorbyl phosphate (VCP-IS-Na); Tetraisopalmitoyl ascorbic acid (TIPA); Ascorbic acid sulfate; Coenzyme Q10 (Ubiquinone); Lactic acid; Ferulic Acid; Hydrolyzed collagen + hyaluronic acid (Cromoist HTA); Bovine raw milk exosomes; Bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate); MSC exosomes; MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate); and any combination thereof.

[0088] By way of non-limiting example, one or more exemplary embodiments of a composition comprise a vanishing base cream (supplied from commercial source; such as Humco PENcream™ or Humco MultiBase™); Vitamin A (as retinol) 2.5g/100g; Vitamin B3 5 g/10Og; Vitamin E 50,000IU/100g; Vitamin C (as Tetrahexyldecyl Ascorbate (THD Ascorbate); Magnesium ascorbyl phosphate (MAP); Ascorbyl 6 palmitate; Disodium isostearyl 2-0 L-ascorbyl phosphate (VCP-IS- Na); Tetraisopalmitoyl ascorbic acid (TIPA); Ascorbic acid sulfate) 15-20g/100g; Coenzyme Q10 (Ubiquinone) 3-6%; Lactic acid 1-5%; Ferulic Acid 0.8-1%; Hydrolyzed collagen + hyaluronic acid (Cromoist HTA) 2g/100g; Bovine raw milk exosomes (1E13 EVs/ml); Bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) 50X concentrate; MSC exosomes (1E12 EVs/ml); MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate; and any combination thereof. [0089] Exemplary methods for preparing the disclosed anti-wrinkle and brightening eye compositions are herein provided. One of ordinary skill in the art, having the benefit of the present disclosure, will appreciate that alternative methods may be available without departing from the spirit and scope of the present disclosure.

[0090] Exemplary Method 4.1

[0091] One or more embodiments of the present disclosure comprise at least one of (i) selecting a vanishing base cream from a commercial source such as Humco PENcream™ or Humco MultiBase™ (ii) providing 5g of the base cream at ambient temperature; (iii) adding Vitamin A (as retinol) 0.625g/25g, Vitamin B3 1.25 g/25g, E 12,500IU/25g, Hydrolyzed collagen + hyaluronic acid (Cromoist HTA) 0.5g/25g and mix at 50 rpm in 45 ^o C for 10 min; (iv) adding the following: (adjust concentrations and amounts as per need) Vitamin C (as Tetrahexyldecyl Ascorbate (THD Ascorbate); Magnesium ascorbyl phosphate (MAP); Ascorbyl 6 palmitate; Disodium isostearyl 2-0 L-ascorbyl phosphate (VCP-IS-Na); Tetraisopalmitoyl ascorbic acid (TIPA); Ascorbic acid sulfate) 3.75-5g/25g; (v) adding Coenzyme Q10 (Ubiquinone) 0.75- 1.5g/25g; (vi) adding a mixture of Lactic acid 1-5% and Ferulic acid 0.2-0.25g/25g; (vii) adding 0.5 ml of bovine raw milk exosomes (1E13 EVs/ml); (viii) adding 0.5ml of bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) 50X concentrate; (ix) adding 1ml of MSC exosomes (1E12 EVs/ml); (x) adding 1ml of MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate– 3 KDa retentate) 50X concentrate; (xi) mixing at 35 rpm in 16 ^o C for 10 min; and (xii) adjusting to 10g using base cream.

[0092] Exemplary Embodiment 5

[0093] One or more embodiments of the present disclosure provide a composition for facial skin rejuvenation comprising at least one of umbilical cord or other mesenchymal stem cells (MSC) and milk exosome. One or more embodiments of a composition may comprise a vanishing base cream (supplied from commercial source; such as Humco PENcream™ or Humco MultiBase™); Vitamin A (as retinol); Vitamin B3; Vitamin E; Bovine raw milk exosomes; Bovine raw milk Ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate); Umbilical Cord MSC exosomes; Umbilical Cord MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate– 3 KDa retentate); and any combination thereof. [0094] By way of non-limiting example, one or more embodiments of a composition may comprise a vanishing base cream (supplied from commercial source; such as Humco PENcream™ or Humco MultiBase™); Vitamin A (as retinol) 2.5g/100g; Vitamin B3 5 g/10Og; Vitamin E 50,000IU/100g; Bovine raw milk exosomes (5E13 EVs/ml); Bovine raw milk Ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) 50X concentrate; Umbilical Cord MSC exosomes (5E12 EVs/ml); Umbilical Cord MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate; and any combination thereof.

[0095] Exemplary methods for preparing the disclosed facial skin rejuvenation compositions are herein provided. One of ordinary skill in the art, having the benefit of the present disclosure, will appreciate that alternative methods may be available without departing from the spirit and scope of the present disclosure.

[0096] Exemplary Method 5 1

[0097] One or more embodiments of the present disclosure may comprise at least one of (i) performing the steps according to at least one of exemplary methods 3.1, 3.2, and 3.3 described hereinabove; and (ii) adding 1ml of bovine raw milk exosomes (5E13 EVs/ml); 0.5ml of bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) 50X concentrate; 1ml of Umbilical Cord MSC exosomes (5E12 EVs/ml); 1ml of Umbilical Cord MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate, and any combination thereof; (iii) mixing at 35 rpm at 16°C for 10 min; and (iv) adjusting to 20g using base cream.

[0098] Exemplary Composition 6

[0099] Exosomes released by mouse mast cells exposed to oxidative stress differ in their mRNA content. Exosomes can influence the response of other cells to oxidative stress by providing recipient cells with a resistance against oxidative stress, observed as an attenuated loss of cell viability. Exposure to UV-light affects the biological functions associated with exosomes released under oxidative stress which happens also to the skin cells. Exosomes from bone marrow-derived mesenchymal stem cells (BMSCs) facilitate cell proliferation and survival by transporting various bioactive molecules, including microRNAs (miRs). BMSC-derived exosomes significantly decrease apoptosis rates and reactive oxygen species (ROS) production. A stronger effect is induced by exosomes collected from BMSCs cultured under hypoxic conditions than those collected from BMSCs cultured under normal conditions. Higher miR-214 enrichment in Hypoxic exosomes than in Normoxic exosomes. Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate P13/Akt pathway.

[00100] However, the antioxidants released from astrocytes either via extracellular fluid or exosomes to neurons during stress conditions may not be sufficient to provide neuroprotection. Therefore, to combat oxidative stress in the central nervous system, synthetically developed exosomes loaded with antioxidants such as glutathione and the anti-aging protein Klotho may be employed.

[00101] Oxidative stress induction was simulated using a bioreactor to change the dissolved oxygen (DO%) in the medium. Normal dissolved oxygen (DO%), which ranges from about 30- 50% in normoxic culture, was adjusted at 80-100% in oxidative stress induction conditions for varying time periods to evaluate the expression profile in the resulting exosomes, while all other process parameters were maintained at their optimum values. Downstream processes remained the same and the final exosomal and secretome were incorporated into the cosmeceutical preparation.

[00102] Accordingly, one or more embodiments of the present disclosure provide a composition for anti-aging (antioxidants, aka Serum) comprising retinols; milk exosomes and peptides (collagen stimulants); A-MSC exosomes under hyperbaric O ₂ ; and any combination thereof.

[00103] By way of non-limiting example, one or more embodiments of the present disclosure may comprise a vanishing base cream (supplied from commercial source; such as Humco PENcream™ or Humco MultiBase™); Vitamin A (as retinol); Vitamin E; Bovine raw milk exosomes; Bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate); A-MSC exosomes at hyperbaric O ₂ conditions; A-MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) at hyperbaric O ₂ conditions; and any combination thereof.

[00104] One or more embodiments of the present disclosure comprise a vanishing base cream (supplied from commercial source; such as Humco PENcream™ or Humco MultiBase™); Vitamin A (as retinol) 2.5g/100g; Vitamin E 50,000IU/100g; Bovine raw milk exosomes (5E13 EVs/ml); Bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) 50X concentrate; A-MSC exosomes (5E12 EVs/ml) at hyperbaric O ₂ conditions; A-MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate at hyperbaric O ₂ conditions; and any combination thereof.

[00105] Exemplary methods for preparing the disclosed anti-aging compositions are herein provided. One of ordinary skill in the art, having the benefit of the present disclosure, will appreciate that alternative methods may be available without departing from the spirit and scope of the present disclosure.

[00106] Exemplary Method 6 1

[00107] One or more embodiments of the present disclosure may comprise at least one of (i) performing the steps according to at least one of exemplary methods 3.1, 3.2, and 3.3 described hereinabove; (ii) adding 1ml of bovine raw milk exosomes (5E13 EVs/ml), 0.5ml of bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) 50X concentrate, 1ml of A-MSC exosomes (5E12 EVs/ml) at hyperbaric O ₂ conditions, 1ml of A-MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate at hyperbaric O ₂ conditions; (iii) mixing at 35 rpm at 16°C for 10 min; and adjusting to 20g using base cream.

[00108] Exemplary Embodiment 7

[00109] One or more embodiments of the present disclosure provide a composition for wound healing cream comprising platelet-rich plasma (PRP); induced pluripotent stem cells (iPCs); MSC exosomes; O/W cream; and any combination thereof.

[00110] One or more embodiments of the present disclosure comprise a vanishing base cream (supplied from commercial source; such as Humco PENcream™ or Humco MultiBase™); Vitamin A (as retinol); Vitamin E; PRP isolated exosomes; iPCs isolated exosomes; A-MSC exosomes at hyperbaric O ₂ conditions; A-MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) at hyperbaric O ₂ conditions; and any combination thereof. [00111] One or more embodiments of the present disclosure comprise a vanishing base cream (supplied from commercial source; such as Humco PENcream™ or Humco MultiBase™); Vitamin A (as retinol) 2.5g/100g; Vitamin E 50,000IU/100g; PRP isolated exosomes; iPCs isolated exosomes; A-MSC exosomes (5E12 EVs/ml) at hyperbaric O ₂ conditions; A-MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate at hyperbaric O ₂ conditions; and any combination thereof.

[00112] Exemplary methods for preparing the disclosed wound healing compositions are herein provided. One of ordinary skill in the art, having the benefit of the present disclosure, will appreciate that alternative methods may be available without departing from the spirit and scope of the present disclosure.

[00113] Exemplary Method 7 1

[00114] One or more embodiments of the present disclosure may comprise at least one of (i) performing the steps according to at least one of exemplary methods 3.1, 3.2, and 3.3 described hereinabove; (ii) adding 1ml of bovine raw milk exosomes (5E13 EVs/ml), 1ml of A-MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate at hyperbaric O ₂ conditions, PRP isolated exosomes, iPCs isolated exosomes; (iii) and mixing at 35 rpm at 16°C for 10 min; and (iv) adjusting to 20g using base cream.

[00115] Exemplary Embodiment 8

[00116] Hyperbaric CO ₂ induced MSC exosomes (angiogenic factors) MSC exosomes produced in hypoxia situation show different miRNA and protein profiles. The angiogenesis related factors are over-expressed in which different signaling pathways are involved including nuclear factor- kappaB signaling. Several putative paracrine effectors of angiogenesis present in MSC exosomes and increased in expression in MSCs exposed to hypoxia that include platelet derived growth factor, epidermal growth factor, overexpressing Nrf2, fibroblast growth factor, and most notably nuclear factor-kappaB (NFkB) signaling pathway proteins.

[00117] Overexpression of angiogenesis-related factors may be useful in stimulating vascularization in diabetic patients suffering from diabetic foot ulcer, which is caused by the effects of chronic ischemia, typically due to peripheral artery disease. These angiogenesis-related factors may stimulate pericytes to vascularize the affected tissue.

[00118] The hyperbaric CO ₂ and/or hypoxia conditions were simulated using the bioreactor to change the CO ₂ % and/or dissolved oxygen (DO%) in the medium. Normal CO ₂ % range is typically about 5-10% during the experiments and/or dissolved oxygen (DO%) which ranged between about 30-50% in normoxic culture was adjusted to about 1-20% in hypoxic conditions. For the hyperbaric CO ₂ induction, a range of aboutl0-40% was applied for varying time periods to evaluate the expression profile in the resulting exosomes, while all other process parameters were maintained at their optimum values. Downstream processes remained the same and the final exosomal and secretome was incorporated into the cosmeceutical preparation.

[00119] Accordingly, one or more embodiments of the present disclosure provide a composition for diabetic wound treatment comprising hyperbaric CO ₂ induced MSC exosomes having at least one angiogenic factor and/or Nrf2 and any combination thereof.

[00120] One or more non-limiting embodiments may comprise a vanishing base cream (supplied from commercial source; such as Humco PENcream™ or Humco MultiBase™); Vitamin A (as retinol); Vitamin E; A-MSC exosomes at hyperbaric CO ₂ or hypoxia conditions; A-MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) at hyperbaric O ₂ conditions at hyperbaric CO ₂ or hypoxia conditions; and any combination thereof.

[00121] One or more non-limiting embodiments may comprise a vanishing base cream (supplied from commercial source; such as Humco PENcream™ or Humco MultiBase™); Vitamin A (as retinol) 2.5g/100g; Vitamin E 50,000IU/100g; A-MSC exosomes (5E12 EVs/ml) at hyperbaric CO ₂ or hypoxia conditions; A-MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate at hyperbaric O ₂ conditions at hyperbaric CO ₂ or hypoxia conditions; and any combination thereof.

[00122] Exemplary methods for preparing the disclosed diabetic wound treatment compositions are herein provided. One of ordinary skill in the art, having the benefit of the present disclosure, will appreciate that alternative methods may be available without departing from the spirit and scope of the present disclosure. [00123] Exemplary Method 8.1

[00124] One or more embodiments of the present disclosure may comprise at least one of (i) performing the steps according to at least one of exemplary methods 3.1, 3.2, and 3.3 described hereinabove; (ii) adding 1ml of bovine raw milk exosomes (5E13 EVs/ml); 0.5ml of bovine raw milk ultracentrifugation peptide/protein fraction or 750 KDa TFF filtrate (100 KDa retentate) 50X concentrate; 1ml of A-MSC exosomes (5E12 EVs/ml) at hyperbaric O ₂ conditions; 1ml of A-MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate at hyperbaric O ₂ conditions; (iii) mixing at 35 rpm at 16°C for 10 min; and (iv) adjusting to 20g using base cream.

[00125] Exemplary Embodiment 9

[00126] One or more embodiments of the present disclosure provide a composition for treating atopic dermatitis comprising bone marrow MSC exosomes.

[00127] By way of non-limiting example, one or more embodiments of the present disclosure comprise a vanishing base cream, such as Humco PENcream™; Bone Marrow MSC exosomes; Bone Marrow MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate); and any combination thereof.

[00128] In one or more embodiments, a composition comprises a vanishing base cream, such as Humco PENcream™; Bone Marrow MSC exosomes (1E13 EVs/ml); Bone Marrow MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate; and any combination thereof.

[00129] Exemplary methods for preparing the disclosed atopic dermatitis compositions are herein provided. One of ordinary skill in the art, having the benefit of the present disclosure, will appreciate that alternative methods may be available without departing from the spirit and scope of the present disclosure.

[00130] Exemplary Method 9 1

[00131] One or more embodiments of the present disclosure may comprise at least one of (i) performing the steps according to at least one of exemplary methods 3.1, 3.2, and 3.3 described hereinabove; (ii) adding 1ml of Bone Marrow MSC exosomes (1E13 EVs/ml); (iii) adding 1ml of Bone Marrow MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate; (iv) mixing at 35 rpm in 16°C for 10 min; and (v) Adjust to 50g using base cream.

[00132] Exemplary Embodiment 10

[00133] One or more embodiments of the present disclosure provide a lip cream composition comprising at least one of umbilical cord MSC exosomes and umbilical cord MSC secretome.

[00134] By way of non-limiting example, one or more embodiments comprise a highlighter balm; Umbilical cord MSC exosomes; Umbilical cord MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate); and any combination thereof.

[00135] One or more exemplary embodiments may comprise a highlighter balm (1445, for example); Umbilical cord MSC exosomes (5E12 EVs/ml); Umbilical cord MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate; and any combination thereof.

[00136] Exemplary methods for preparing the disclosed day/night lip cream compositions are herein provided. One of ordinary skill in the art, having the benefit of the present disclosure, will appreciate that alternative methods may be available without departing from the spirit and scope of the present disclosure.

[00137] Exemplary Method 10.1

[00138] One or more embodiments of the present disclosure may comprise at least one of (i) performing the steps according to at least one of exemplary methods 3.1, 3.2, and 3.3 described hereinabove; (ii) adding 1ml of Bone Marrow MSC exosomes (1E13 EVs/ml); (iii) adding 1ml of Bone Marrow MSC secretome (MSC culture conditioned medium; TFF 750 KDa filtrate - 3 KDa retentate) 50X concentrate; and (iv) adjust to 50g using base cream.

[00139] In addition to the disclosed embodiments, one or more additional embodiments of the present disclosure may comprise preparing and/or providing one or more of the above-referenced compositions and applying said composition to the skin of an individual. Moreover, one or more embodiments may comprise preparing and/or providing one or more of the above-referenced compositions and instructing a person to apply the composition in accordance with a predefined treatment protocol, wherein the person applies the composition in accordance with said protocol.

[00140] Although particular embodiments have been disclosed herein in detail, this has been done for purposes of illustration only, and is not intended to be limiting with respect to the scope of any claims. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made without departing from the spirit and scope of the embodiments described, including any variation in the amount and/or concentration of a component of the various embodiments.

EXAMPLES

[00141] Microbial challenge test

[00142] A microbial challenge test was performed on an exemplary exosome-based anti-aging rejuvenating cream to evaluate the effectiveness of antimicrobial preservatives against microbial contamination. The exemplary embodiment comprised a base cream (Humco PENcream™) and A-MSC exosomes. The method employed was USP 42, Section 51, Antimicrobial Effectiveness Testing, as is known in the art.

[00143] Test organisms included Staphylococcus aureus ATCC#6538; Escherichia coli ATCC#8739; Pseudomonas aeruginosa ATCC#9027; Candida albicans ATCC# 1023, and Aspergillus brasiliensis ATCC# 16404.

[00144] RESULTS

[00145] Table 2. Microbial Challenge Test Results (Log10 CFU/g or CFU/ml)

[00146] Table 3. Suitability of Counting Method Validation*

* Suitability of the counting method is confirmed when recovery of the test organisms in the sample is at least 50% of that of the control. Test product dilutions past 10 ^-1 not reported where acceptable recovery is demonstrated on the 10 ^-1 dilution.

[00147] ACCEPTANCE CRITERIA

[00148] For category 2 products, the preservative is effective in the sample examined if a) the concentrations of viable bacteria demonstrate no less than a 2.0 log reduction from the initial count at 14 days and no increase from the day 14 count at 28 days; and b) the concentrations of viable yeast and molds demonstrate no increase from the initial calculated count at 14 and 28 days.

[00149] CONCLUSION

[00150] The test material, Exosome-based anti-aging rejuvenating cream, conforms to the acceptance criteria for USP 42 <51> category 2 products.

[00151] Western Blot Characterization of Exosomes

[00152] Exosomes were characterized by standard western blots using techniques known in the art, and as indicated in Table 4 below. The western blots demonstrate the presence of typical exosome biomarkers, thereby validating the exosome isolation techniques employed in connection with the embodiments disclosed herein. The results of this characterization are disclosed in FIGS. 1A-4C. [00153] Table 4. Western Blot Criteria

[00154] Pyrogenic materials

[00155] Lipopolysaccharide (LPS) content was measured using techniques known in the art.

[00156] LIPIDOMICS

[00157] Sample type: A-MSC Exosome

Extraction Method: Bligh and Dyer

Internal Standards: SPLASH + Lipidyzer

LC: Shimadzu LC system

MS Instrument: Linear Ion Trap Quadrupole LC/MS/MS Mass Spectrometer

Model: QTRAP 6500

Company: AB SCIEX Instruments

Scan Type: MRM (Scheduled)

Polarity: Positive/Negative Injection volume: 5 uL

Unit: mM

LC Column: 1-Amide Column: XBridge Amide 3.5 mih, 4.6x 150 mm column (Waters, Ireland) Mobile phase A: Acetonitrile: Water (95:5) with ImM Ammonium acetate, pH=8.2

Mobile phase B: Acetonitrile: Water (50:50) with ImM Ammonium acetate, pH=8.2

Data explanation:

1- 20 mL exosome was used for lipid extraction.

2-Lipids were quantified semi-quantitatively. Data were first normalized based on internal standards and concentrations were calculated based on a surrogate internal standard.

Lipopolysaccharides: Not detected

[00158] Lipid content of A-MSC exosomes

[00159] Lipid content was measured under the following criteria.

[00160] LIPIDOMICS

Sample type: A-MSC Exosome

Extraction Method: Bligh and Dyer

Internal Standards: SPLASH + Lipidyzer

LC: Shimadzu LC system

MS Instrument: Linear Ion Trap Quadrupole LC/MS/MS Mass Spectrometer

Model: QTRAP 6500

Company: AB SCIEX Instruments

Scan Type: MRM (Scheduled)

Polarity: Positive/Negative

Injection volume: 5 uL

Unit: mM LC Column: 1-Amide Column: XBridge Amide 3.5 mih, 4.6x 150 mm column (Waters, Ireland) Mobile phase A: Acetonitrile: Water (95:5) with ImM Ammonium acetate, pH=8.2

Mobile phase B: Acetonitrile: Water (50:50) with ImM Ammonium acetate, pH=8.2

Data explanation:

1- 20 mL exosome was used for lipid extraction.

2-Lipids were quantified semi-quantitatively. Data were first normalized based on internal standards and concentrations were calculated based on a surrogate internal standard.

[00161] Table 5. Lipid Content Results for A-MSC Exosomes.

Concentration

Lipid Species

(mM)

SM(14:0)+H 0.07213

SM(16:0)+H 0.71884

SM(18:0)+H 0.04687

SM(18:1)+H 0.01309

SM(20:0)+H 0.01032

SM(20:1)+H 0.00299

SM(22:0)+H 0.05655

SM(22:1)+H 0.02795

SM(24:0)+H 0.11129

SM(24:1)+H 0.31208

SM(26:0)+H 0.00285

SM(26:1)+H 0.00609

CE(24:0)+H 1.91129

CE(14:0)+H 16.44606

CE(18:3)+H 3.70328

CE(20:2)+H 1.66904

CE(22:0)+H 4.06422

CE(22: 1)+H 6.46284

CER(14:0)+H 0.62088

CER(16:0)+H 0.02152

CER(22:0)+H 0.01057

CER(24: 1)+H 0.05315

DCER(14:0)+H 0.02842

DCER(20:0)+H 0.00891

DCER(20: 1)+H 0.01992 DCER(22:0)+H 0.00335

DCER(22: 1)+H 0.00128

DCER(24:0)+H 0.02728

DCER(24: 1)+H 0.00584

HCER(18: 1)+H 0.00028

HCER(20: 1)+H 0.00038

HCER(22: 1)+H 0.00033

HCER(24:0)+H 0.00015

HCER(24: 1)+H 0.00050

HCER(26:0)+H 0.00007

HCER(26: 1)+H 0.00011

HCER(d 18:0/20: 0)+H 0.00013

T AG(44 : 2/FA 16 : 1 )+NH4 0.03816

TAG(45: 1/FA18: 1)+NH4 0.02162

TAG(47:2/FA18: 1)+NH4 0.08057

T AG(48 : 0/F A 18 : 0)+NH4 0.06360

T AG(48 : 2/FA 18 : 2)+NH4 0.02968

TAG(48:4/FA20:4)+NH4 0.05512

TAG(49: 1/FA16:0)+NH4 0.09150

TAG(49: 1/FA17:0)+NH4 0.04071

T AG(49 : 2/FA 18 : 1 )+NH4 0.22205

TAG(49:2/F A18 :2)+NH4 0.09552

TAG(49:3/FA18:2)+NH4 0.19554

TAG(50: 1/FA18:0)+NH4 0.08904

TAG(50: 1/FA18: 1)+NH4 0.06458

TAG(50: 1/FA20: 1)+NH4 0.05048

T AG(50 : 2/FA 18 : 1 )+NH4 0.11208

TAG(50:2/FA18:2)+NH4 0.06785

TAG(50:3/FA18:2)+NH4 0.12883

TAG(50:4/FA18: 1)+NH4 0.06785

TAG(50:4/FA18:3)+NH4 0.05863

TAG(50:5/FA14:0)+NH4 0.06881

TAG(50:5/FA18:2)+NH4 0.04411

TAG(50:5/FA18:3)+NH4 0.02969

TAG(50:5/FA20:5)+NH4 0.06785

T AG(51 : 0/F A 17 : 0)+NH4 0.07944

TAG(51 : 1/FA16:0)+NH4 0.07615

TAG(51 : 1/FA17:0)+NH4 0.79503

TAG(51 : 1/FA18:0)+NH4 0.01620

TAG(51 : 1/FA18: 1)+NH4 0.05196

T AG(51 : 2/FA 16 : 0)+NH4 0.27344 T AG(51 : 2/FA 16 : 1 )+NH4 0.02286 TAG(51 :2/FA18: 1)+NH4 0.20526 TAG(51 :2/F A18 :2)+NH4 0.03529 TAG(51 :3/FA16: 1)+NH4 0.02544 TAG(51 :4/F A18 :3)+NH4 0.02400 TAG(52: 1/FA20:0)+NH4 0.07916 T AG(52 : 2/FA 16 : 0)+NH4 0.03179 T AG(52 : 2/FA 18 : 1 )+NH4 0.05192 T AG(52 : 2/FA 18 : 2)+NH4 0.00666 T AG(52 : 3/F A14 : 0)+NH4 0.11646 T AG(52 : 3/F A18 : 1 )+NH4 0.07688 TAG(52:3/FA18:2)+NH4 0.05766 TAG(52:3/FA20: 1)+NH4 0.03089 T AG(52 : 3/F A20 :2)+NH4 0.04207 T AG(52 : 4/FA 16 : 1 )+NH4 0.02831 T AG(52 : 4/FA 18 : 2)+NH4 0.04908 TAG(52:4/FA20:4)+NH4 0.03398 T AG(52 : 5/F A20 : 5)+NH4 0.02161 T AG(52 : 6/F A20 : 5 )+NH4 0.01179 T AG(52 : 8/F A 18 : 2)+NH4 0.01966 TAG(53 : 1/FA17:0)+NH4 0.33342 TAG(53 :2/FA17:0)+NH4 0.44098 TAG(53 :2/F A18 : 1)+NH4 0.08822 TAG(53 :3/FA17:0)+NH4 0.55277 TAG(53 :4/FA17:0)+NH4 0.16591 TAG(53 :4/F A18 :2)+NH4 0.15979 TAG(53 :6/FA20:4)+NH4 0.01178 TAG(54: 1/FA18: 1)+NH4 0.08254 T AG(54 : 2/FA 18 : 1 )+NH4 0.04323 TAG(54:2/FA20:2)+NH4 0.14525 T AG(54 : 3/F A18 : 0)+NH4 0.01296 T AG(54 : 3/F A 18 : 1 )+NH4 0.09068 T AG(54 : 4/FA 18 : 1 )+NH4 0.03851 T AG(54 : 5/F A 18 : 1 )+NH4 0.01418 TAG(54:5/FA18:2)+NH4 0.01032 T AG(54 : 6/F A 18 : 1 )+NH4 0.00838 T AG(54 : 6/F A 18 : 2)+NH4 0.01640 T AG(54 : 6/F A20 : 4)+NH4 0.01032 T AG(54 : 6/F A22 : 6)+NH4 0.01592 TAG(54:7/FA18:3)+NH4 0.00789 TAG(54:7/FA22:6)+NH4 0.02672 T AG(54 : 8/FA 18 : 2)+NH4 0.00972

T AG(55 : 2/FA 18 : 1 )+NH4 0.00486

TAG(55:5/FA18: 1)+NH4 0.02837

TAG(56:3/FA18: 1)+NH4 0.02282

TAG(56:4/FA20: 1)+NH4 0.00208

TAG(56:4/FA20:2)+NH4 0.00092

T AG(56 : 6/FA 18 : 0)+NH4 0.00424

T AG(56 : 6/F A22 : 5 )+NH4 0.00318

TAG(56:7/FA18:2)+NH4 0.00393

T AG(58 : 6/F A20 : 4)+NH4 0.00092

T AG(58 : 6/F A22 : 5)+NH4 0.00162

TAG(60: 1 1/FA22:6)+NH4 0.00190

DAG(14:0/14:0)+NH4 I .07354

DAG(14:0/16: 1)+NH4 0.35656

DAG(16:0/16:0)+NH4 0.44489

DAG(16:0/16: 1)+NH4 0.20687

DAG(14:0/18: 1)+NH4 I I .56397

DAG(16: 1/16: 1)+NH4 0.32172

D AG( 14:0/18: 2)+NF44 11.76252

DAG(16: 1/18:0)+NH4 0.86065

DAG(16:0/18: 1)+NH4 7.43409

DAG(16: 1/18: 1)+NH4 25.01781

D AG( 16: 1/18: 2)+NH4 0.81 1 19

DAG(16:0/18:3)+NH4 0.48630

DAG(16: 1/18:3)+NH4 0.30584

DAG(14:0/20:4)+NH4 0.48265

DAG(16: l/20:0)+NH4 0.54642

DAG(18:0/18: 1)+NH4 0.53764

DAG(1B: 1/18: 1)+NH4 1.56569

D AG( 18:0/18: 2)+NF44 1.18960

DAG(1B: 1/18:2)+NH4 0.28234

DAG(18:0/18:3)+NH4 0.40922

D AG( 16: 1/20: 2)+NF44 0.45044

DAG(16:0/20:3)+NH4 0.12535

D AG( 16:0/20: 4)+NF44 0.12352

D AG( 16: 1/20: 4)+NF44 0.35612

DAG(16:0/20: 5)+NH4 0.06973

D AG( 14:0/22: 6)+NF44 0.12015

D AG( 18:2/20: 5)+NH4 0.48798

D AG( 18: 1/22: 4)+NF44 0.31248

D AG( 18:0/22: 6)+NF44 12.80632 M AG( 16 : 0)+NH4 1.77107 M AG( 16: 1 )+NH4 2.15081 M AG( 18 : 0)+NH4 16.11039 M AG( 18: 1 )+NH4 17.46537 M AG( 18 : 2)+NH4 2.54008 M AG(20 : 0)+NH4 1.88197 M AG(20 : 1 )+NH4 1.94088 M AG(20 : 2)+NH4 1.77442 MAG(20:3)+NH4 2.21464 M AG(20 : 4)+NH4 1.43079 M AG(22 : 0)+NH4 0.80881 M AG(22 : 1 )+NH4 0.90217 M AG(22 : 2)+NH4 1.25383 M AG(22 : 3 )+NH4 1.40984 M AG(22 : 4)+NH4 0.60677 MAG(22:5)+NH4 1.50153 MAG(22:6)+NH4 1.26461 LPC(16:0)+AcO 0.01471 LPC(16: l)+AcO 0.00329 LPC(18:0)+AcO 0.02612 LPC(18: l)+AcO 0.01745 LPC(18:2)+AcO 0.01966 LPC(20:0)+AcO 0.21392 LPC(20:4)+AcO 0.03161 LPC(20:5)+AcO 0.00890 PC(16:0/20:4)+AcO 0.31803 PC(16:0/22:6)+AcO 0.11441 PC(18:0/18:0)+AcO 0.46047 PC(18:0/20:0)+AcO 1.14246 PC(18:0/20:3)+AcO 0.16399 PC(18:0/20:4)+AcO 0.49047 PC(18: 0/22:5 )+ AcO 0.12330 PC(18:0/22:6)+AcO 0.19950 PC(18: 1/20:4)+ AcO 0.13123 PC(18: 1/22:6)+ AcO 0.04596 PC(20 : 0/20 :4)+ AcO 0.04276 PC(20: 0/22:6)+ AcO 0.02643 LPE(16:0)-H 0.00173 LPE(16: 1)-H 0.00026 LPE(18:0)-H 0.00104 LPE(18: 1)-H 0.00119 LPE(18:2)-H 0.00261

LPE(18:3)-H 0.00027

LPE(20:0)-H 0.00006

LPE(20: 1)-H 0.00015

LPE(20:2)-H 0.00016

LPE(20:3)-H 0.00228

LPE(20:4)-H 0.00486

LPE(20:5)-H 0.00163

LPE(22:4)-H 0.00012

PE(14:0/18: 1)-H 0.00171

PE(16:0/18: 1)-H 0.03411

PE(16:0/18:2)-H 0.00181

PE(16:0/20: 1)-H 0.00077

PE(16:0/20:2)-H 0.00127

PE(16:0/20:3)-H 0.00331

PE(16:0/20:4)-H 0.00304

PE(16:0/22:4)-H 0.00499

PE(16:0/22:5)-H 0.00284

PE(16:0/22:6)-H 0.00067

PE(18:0/16:0)-H 0.00136

PE(18:0/16: 1)-H 0.00276

PE(18:0/18:0)-H 0.00385

PE(18:0/18: 1)-H 0.08735

PE(18:0/18:2)-H 0.00751

PE(18:0/20: 1)-H 0.00173

PE(18:0/20:2)-H 0.00556

PE(18:0/20:3)-H 0.02056

PE(18:0/20:4)-H 0.02382

PE(18:0/22:4)-H 0.01556

PE(18:0/22:5)-H 0.00432

PE(18: 1/16: 1)-H 0.00354

PE(18: 1/18: 1)-H 0.13849

PE(18: 1/18:2)-H 0.00612

PE(18: 1/20: 1)-H 0.00389

PE(18: l/20:2)-H 0.00520

PE(18: l/20:3)-H 0.01514

PE(18: l/20:4)-H 0.01362

PE(18: l/22:4)-H 0.00796

PE(18: l/22:5)-H 0.00813

PE(0- 16:0/16: 0)-H 0.00266

PE(O-16:0/16: l)-H 0.00200 RE(O-16:0/18: 1)-H 0.00182 PE(0- 16:0/18 :3)-H 0.00095 PE(0- 16:0/20: 4)-H 0.00868 PE(0-16:0/20: 5)-H 0.00380 PE(P- 16:0/16: 0)-H 0.00418 PE(P-16:0/16: 1)-H 0.00458 PE(P- 16:0/18: 0)-H 0.00122 PE(P-16:0/18: 1)-H 0.04969 PE(P- 16:0/18: 2)-H 0.00365 PE(P- 16:0/18 :3)-H 0.00087 PE(P- 16:0/20: 1)-H 0.00917 PE(P- 16:0/20: 2)-H 0.01433 PE(P- 16:0/20:3 )-H 0.04122 PE(P- 16:0/20: 4)-H 0.04492 PE(P-16:0/22:4)-H 0.05504 PE(P-16:0/22: 5)-H 0.39469 PE(P-16: 1/18: 1)-H 0.00062 PE(P- 18:0/16: 0)-H 0.00670 PE(P-18:0/16: 1)-H 0.00416 PE(P- 18:0/18: 0)-H 0.00061 PE(P-18:0/18: 1)-H 0.01650 PE(P- 18:0/18: 2)-H 0.00234 PE(P- 18:0/20: 1 )-H 0.00250 PE(P- 18:0/20: 2)-H 0.00315 PE(P- 18:0/20:3 )-H 0.01315 PE(P- 18:0/20: 4)-H 0.04276 PE(P-18:0/22:4)-H 0.02263 PE(P- 18 : 0/22 : 5 )-H 0.00620 PE(P- 18 : 0/22 : 6)-H 0.00381 PE(P-18: 1/16:0)-H 0.03127 PE(P-18: 1/16: 1)-H 0.00643 PE(P-18: 1/18:0)-H 0.00113 PE(P-18: 1/18: 1)-H 0.03018 PE(P-18: 1/18:2)-H 0.00297 PE(P-18: 1/20: 1)-H 0.00317 PE(P- 18: 1/20: 2)-H 0.00399 PE(P- 18: 1/20:3 )-H 0.02934 PE(P- 18: 1/20: 4)-H 0.08198 PE(P- 18: l/22:4)-H 0.02682 PE(P- 18 : 1/22 : 5)-H 0.02253 PE(P- 18 : 1/22 : 6)-H 0.00262 PG(18:0/18: 1)-H 0.00027

PG(18:0/18:2)-H 0.00004

PG(18: 1/18:2)-H 0.00004

PG(18: l/20:4)-H 0.00003

PG(20:0/18: 1)-H 0.00069

PG(20:0/18:2)-H 0.00062

PG(20:0/18:3)-H 0.00013

PG(20:0/20:3)-H 0.00015

PG(20:0/20:4)-H 0.00047

PG(20:0/20:5)-H 0.00007

PG(20:0/22:5)-H 0.00009

PG(20:0/22:6)-H 0.00017

PI(18:0/20:3)-H 0.00585

PI(18:0/20:4)-H 0.00599

PS(16:0/20:3)-H 0.00020

PS(16:0/22:4)-H 0.00022

PS(16:0/22:6)-H 0.00004

PS(18:0/20:4)-H 0.00011

PS(18: l/20:3)-H 0.00028

PS(18: l/20:4)-H 0.00056

PS(18: l/22:4)-H 0.00023

PS(18: l/22:5)-H 0.00005

PS(18:2/20:3)-H 0.00030

PS(18:2/20:4)-H 0.00050

PS(18:2/22:4)-H 0.00038

[00162] Cell proliferation and cytotoxicity assays

[00163] MTT assay

[00164] The BJ Fibroblasts were seeded at 1E4 cells in a 96-well plate in EMEM and 10% FBS in a final volume of 100 ul/well culture medium in a humidified atmosphere (37°C, 5% CO ₂ ) and penicillin and streptomycin. The HEK 293 cells were seeded at 1E4 cells in a 96-well plate in DMEM and 10% FBS in a final volume of 100 ul/well culture medium in a humidified atmosphere (37°C, 5% CO ₂ ). After two days of culture, A-MSC exosomes and secretome was added to the wells in different concentrations. After different time points, 10 ul of the MTT dye (Sigma Aldrich) was added to each well, and the plate was incubated at 37 °C for 4h in a humidified atmosphere and under 5% CO ₂ . The media content of each well was carefully removed without disturbing cells. Then, 100 ml of DMSO was added to each well and mixed by pipetting up and down. The plate was incubate at 37°C for 15 minutes. After gentle mixing on a shaker for 1 min, the absorbance of the samples against a background control as blank was measured using a microplate reader (BioMate 5, Thermo Spectronic) at 570 nm. The results are depicted in FIGS. 5A-5B.

[00165] WST-1 assays

[00166] The BJ Fibroblasts were seeded at 1E4 cells in a 96-well plate in EMEM and 10% FBS in a final volume of 100 ul/well culture medium in a humidified atmosphere (37°C, 5% CO ₂ ) and penicillin and streptomycin. The HEK 293 cells were seeded at 1E4 cells in a 96-well plate in DMEM and 10% FBS in a final volume of 100 ul/well culture medium in a humidified atmosphere (37°C, 5% CO ₂ ). After two days of culture, A-MSC exosomes and secretome was added to the wells in different concentrations. After different time points, 10 ul of the WST-1 reagent (Roche Applied Science) was added to each well, and the plate was incubated at 37 °C for 2 h in a humidified atmosphere and under 5% CO ₂ . After gentle mixing on a shaker for 1 min, the absorbance of the samples against a background control as blank was measured using a microplate reader (BioMate 5, Thermo Spectronic) at 450 nm. The results are depicted in FIGS. 6A-6D.

[00167] The results show that different concentration of exosomes and secretome have different effects on the BJ fibroblasts and HEK 293 cells. A concentration dependent cell proliferation and viability effect is observed in the BJ fibroblasts and HEK 293 cells. The lower concentrations of exosomes and secretome did not produce significant improvement in cell proliferation in contrast to E10 concentration. Similar trend is observed in both cell types. However, MTT and WST-1 assays resulted in similar viability profiles. The WST-1 assay shows that the enzymatic activity of the BJ fibroblasts improves by 24.9% in 24 hours while the effect is 62.1% in 48 hours. Similar pattern is observed in HEK 293 cells with 40% after 24 hours against 54.5% in 48 hours.

[00168] Wound healing effect (scratch assay)

[00169] The BJ Fibroblasts were seeded at 5E4 cells in a 24-well plate in EMEM and 10% FBS in a final volume of 1 ml/well culture medium and penicillin and streptomycin in a humidified atmosphere (37°C, 5% CO ₂ ). After two days of culture, the plates were exposed to UV light at 365 nm for 10 or 30 minutes using a mini transilluminator machine (BioRad). Then, A-MSC exosomes and secretome was added to the wells in different concentrations. Then a straight scratch was made in each well using a sterile 200 ml pipette tip. At different time points, images of wound scratch were taken under a microscope (Fluorescent, Inverted EVOS FL Life Technologies (Evos)). And scratch areas were analyzed by using Image J (NIH, USA). The results are depicted in FIG. 7

[00170] Compared to control cells, the migration of fibroblasts into scratched areas of monolayers in the presence of 1E8, 1E9 and 1E10 A-MSC exosomes and 50 ul secretome (40X concentrated) increased by 21.8%, 20.8% and 62.1% after 24 h, respectively.